Advantages of crude lysate-based cell-free protein biosynthesis over cell–based protein expression include shorter processing times and the ability to prepare lysates in advance so that they can be stored for as long as possible before starting protein production. Crude cell lysates contain essential components for translation, protein folding, and energy metabolism. Almost any protein encoded by an RNA template can be synthesized by adding amino acids, energy substrates, nucleotides, and salts. In coupled transcription-translation systems, recombinant proteins can be synthesized in a single batch process after the addition of an appropriate RNA polymerase and a DNA template containing an expression cassette with the corresponding promoter. In contrast to cell-based expression methods, cell-free expression results in limited hydrolysis and has the ability to express difficult-to-produce or even toxic proteins or proteins containing specific chemical groups or unnatural amino acids at defined sites. In addition, the open nature of the cell-free expression system allows reactions to be directly controlled and monitored.

ALiCE® Cell-free Expression Technology

ALiCE® cell-free expression platform is developed based on tobacco BY-2 cells. Tobacco BY-2 suspension cells are easy to prepare in a large scale, which provides sufficient cell material for lysate preparation. Most proteins are expressed and accumulate in the BY-2 lysate cytosolic fraction. The BY-2 lysate contains actively-translocating microsomes generated by the disruption of the endoplasmic reticulum during lysate preparation. Thus, protein products can be targeted to microsomal vesicles by containing N-terminal signal peptides, enabling the formation of disulfide bonds and the efficient folding and assembly of complex and polymeric proteins such as enzymes, full-size antibodies, and even membrane proteins. The BY-2 lysate system also supports glycosylation, which is only possible in wheat germ extracts with additional microsomes. The yield of ALiCE® expression system based on BY-2 lysate has been increased from 0.27 mg/mL to 3 mg/mL by optimizing lysate preparation and reaction conditions and extending the transcription-translation process to 48 hours.

Difficult to produce proteins that can be synthesized by the ALiCE® system"Difficult to produce" proteins that can be synthesized by the ALiCE® system

ALiCE® Cell-free Expression Kits

The ALiCE® Cell-free Expression Kit contains all factors required for in vitro transcription (RNA polymerases, NTPs) and translation reactions (ribosomes, translation initiation/elongation factors, tRNA, etc.). Using a simple protocol, both the RNA transcription and translation take place in a single tube. Reactions are started by simply mixing plasmid DNA with the ALiCE® Reaction mix. A reaction duration of 48 h is recommended.

ALiCE® Cell-free Expression Kits are available in three different sizes. Equipped with the ALiCE BY-2 lysate and two expression vectors designed for excellent protein yield, these kits enable researchers to fully control protein production.


ALiCE® Expression Vectors

The two expression vectors in ALiCE® kits are pALiCE01 and pALiCE02, which are used according to the targeting of the protein of interest. These have been specially tailored to offer excellent protein yield when used with the expression system. pALiCE01 and pALiCE02 can also be used as a positive control for expression, expressing eYFP when added to the ALiCE® Reaction mix in the cytosol and the microsomes, respectively.

  • pALiCE01

pALiCE01 is used to express easy-to-produce proteins in cytosolic fraction. Post-translational modifications are not possible with this expression vector.

  • pALiCE02

Targeting the microsomes with pALiCE02 offers the opportunity to express proteins with post-translational modifications like disulfide bonds. Using a melittin signal peptide (MSP), proteins can be translocated into microsomes, where they undergo post-translational modification. Subsequently, the microsomes can be disrupted to recover the expressed proteins.

Map of the pALiCE01 and pALiCE02 vectors and sequence reference pointsMap of the pALiCE01 and pALiCE02 vectors and sequence reference points

Features and Benefits

  • High-yield: up to 3 mg/mL expressed protein
  • Easy to use: one-step reaction protocol
  • Fast: high yield in just 48 hours
  • Flexible: expression of "difficult to produce" proteins, cytotoxic proteins, etc.

Applications

Besides screening, the ALiCE® system can be used for larger scale expression of target proteins. Potential additional applications of the ALiCE® system include, but are not limited to:

  • Target protein characterization
  • Protein optimization
  • Mutant screening
  • Protein-protein interactions
  • Expression analysis
  • Protein localization analysis
  • Protein crystallization and structure analysis
  • Post-translational modification assays
  • Metabolomics
  • Herbicide screening

Kit Contents

  • Mini-Kit
Component Quantity Concentration Volume Storage
ALiCE® Reaction mix 6 n/a 50 µL -80 °C
Vector pALiCE01 1 250 ng DNA/µL 15 µL -20 °C to -80 °C
Vector pALiCE02 1 250 ng DNA/µL 15 µL -20 °C to -80 °C
ALiCE® Tube Caps, perforated 6 n/a n/a Room temp.
  • Midi-Kit
Component Quantity Concentration Volume Storage
ALiCE® Reaction mix 6 n/a 200 µL -80 °C
Vector pALiCE01 1 250 ng DNA/µL 50 µL -20 °C to -80 °C
Vector pALiCE02 1 250 ng DNA/µL 50 µL -20 °C to -80 °C
ALiCE® Tube Set, 12 pcs. 2 n/a n/a Room temp.
  • Maxi-Kit
Component Quantity Concentration Volume Storage
ALiCE® Reaction mix 6 n/a 500 µL -80 °C
Vector pALiCE01 1 250 ng DNA/µL 50 µL -20 °C to -80 °C
Vector pALiCE02 1 250 ng DNA/µL 50 µL -20 °C to -80 °C
ALiCE® Tube Set, 12 pcs. 5 n/a n/a Room temp.

Production of Glucose Oxidase Using the ALiCE® System

ALiCE® was used to produce Aspergillus niger glucose oxidase (GOx), comprising two identical subunits covalently linked by disulfide bonds with eight N-glycosylation sites, without (GOx-His, 65 kDa) and with melittin signal peptide (MSP-GOx-His, 67 kDa). Colorimetric enzyme activity assay (ABTS and HRP) confirmed functionality while glycosylation was proven in immunoblot before (–) and after (+) PNGase F treatment.

Enzyme activity assay and immunoblot assay of GOx expressed in the AliCE® systemEnzyme activity assay and immunoblot assay of GOx expressed in the AliCE® system

Yield Comparison of Protein Expression Systems

Yield comparison of ALiCE® with other cell-free protein expression systemsYield comparison of ALiCE® with other cell-free protein expression systems

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